Sägesser R, Martinez E, Tsagris M, Tabler M
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Crete, Greece.
Nucleic Acids Res. 1997 Oct 1;25(19):3816-22. doi: 10.1093/nar/25.19.3816.
A screening assay for the detection of RNA-binding proteins was developed. It allows the rapid isolation of cDNA clones coding for proteins with sequence-specific binding affinity to a target RNA. For developing the screening protocol, constituents of the human U1 snRNP were utilized as model system. The RNA partner consisted of the U1-RNA stem-loop II and the corresponding protein consisted of the 102 amino acid N-terminal recognition motif of the U1A protein, which was fused to beta-galactosidase and expressed by the recombinant lambda phage LU1A. Following binding of the fusion protein to nitrocellulose membranes, hybridization with a 32P-labeled U1-RNA ligand was carried out to detect specific RNA-protein interaction. Parameters influencing the specificity and the detection limit of binding were systematically investigated with the aid of the model system. Processing the nitrocellulose membranes in the presence of transition metals greatly increased the signal:background ratio. A simple screening protocol involving a single-buffer system was developed. Specific RNA-protein interaction could be detected in the presence of a large excess of recombinant phages from a cDNA library. Only moderate binding affinities (Kd = 10(-8) M) were required. The suitability of the RNA-ligand screening protocol was demonstrated by the identification of new viroid-RNA binding proteins from tomato.
开发了一种用于检测RNA结合蛋白的筛选测定法。它能够快速分离编码对靶RNA具有序列特异性结合亲和力的蛋白质的cDNA克隆。为了制定筛选方案，将人U1 snRNP的成分用作模型系统。RNA伴侣由U1-RNA茎环II组成，相应的蛋白质由U1A蛋白的102个氨基酸的N端识别基序组成，该基序与β-半乳糖苷酶融合并由重组λ噬菌体LU1A表达。融合蛋白与硝酸纤维素膜结合后，与32P标记的U1-RNA配体进行杂交以检测特异性RNA-蛋白质相互作用。借助该模型系统系统地研究了影响结合特异性和检测限的参数。在过渡金属存在下处理硝酸纤维素膜大大提高了信号：背景比。开发了一种涉及单缓冲系统的简单筛选方案。在存在大量来自cDNA文库的重组噬菌体的情况下，可以检测到特异性RNA-蛋白质相互作用。只需要中等的结合亲和力（Kd = 10^(-8) M）。通过从番茄中鉴定新的类病毒-RNA结合蛋白，证明了RNA配体筛选方案的适用性。
